Taxonomic Significance of Pollen Morphology in Succisa and Succisella

Home , Dipsacaceae, Scabiosa, Succisa, Succisa pratensis, Succisella, Succisella inflexa

Biodiv. Res. Conserv. 55: 1-6, 2019 BRC www.brc.amu.edu.pl DOI 10.2478/biorc-2019-0010 Submitted 23.04.2019, Accepted 30.09.2019

Taxonomic significance of pollen morphology in Succisa and Succisella

Zoya M. Tsymbalyuk*, Sergei L. Mosyakin & Lyudmila M. Nitsenko

Department of Systematics and Floristics of Vascular Plants, M. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Tereshchenkivska 2, Kyiv 01004, Ukraine; ORCID: ZMT – https://orcid.org/0000-0003-2768-0045, SLM – https://orcid.org/0000-0002- 3570-3190, LMN – https://orcid.org/0000-0003-1945-7409

*corresponding author (e-mail: [email protected])

Abstract. Pollen morphology of representatives of the genera Succisa and Succisella in the flora of Ukraine was studied using light and scanning electron microscopy. Pollen grains in both taxa were tricolpate, prolate, rarely oblate-spheroidal or spheroidal; large-sized. Their outline in polar view was subcircular, rarely trilobate, in equatorial view elliptical or rarely circular. Colpi short, of variable width, margins irregular with distinct, narrow margo, and blunt or acute ends. Exine sculpture was echinate-microechinate. The revealed characteristics of pollen grains are taxonomically significant at the generic and specific levels, and they can be used in pollen analysis. Palynomorphological data are consistent with the results of recent molecular phylogenetic studies. Data on pollen morphology also confirm taxonomic circumscription of Succiseae V. Mayer & Ehrend. Pollen grains in Succiseae are characterized by a tricolpate aperture type, which was probably ancestral in Dipsacaceae s. str.

Key words: pollen grains, morphology, sculpture, diagnostic characters, taxonomy, Ukraine

1. Introduction a weakly supported sister group to Pseudoscabiosa Devesa (Avino et al. 2009; Carlson et al. 2009; Jacobs Earlier, Succisa Haller and Succisella G. Beck were et al. 2011; Mayer 2016). According to new morpho- usually placed in the family Dipsacaceae Juss. (Bo- logical and molecular phylogenetic studies, Succisa brov 1957; Kotov 1961; Takhtajan 1987, 1997, 2009). and Succisella are included in Succiseae V. Mayer & According to the latest versions of the Angiosperm Ehrend., subfam. Dipsacoideae (Mayer & Ehrendorfer Phylogeny Group (APG III 2009; APG IV 2016) system, 2013; Mayer 2016). they are included in an extended family Caprifoliaceae The genus Succisa, in its current narrow circum- s. l. (also with Diervillaceae Pyck, Morinaceae Raf., scription, includes three species: two with small relict Valerianaceae Batsch, etc.). Molecular phylogenetic ranges (S. pinnatifida Longe in the Iberian Peninsula and studies Dipsacaceae in the strict sense, and of some S. trichotocephala Baksay endemic to W Africa) and related families, were conducted by several groups of one rather widespread species, S. pratensis, naturally researchers (Donoghue et al. 1992; Caputo & Cozzolino occurring in Europe, W Siberia, and N Africa (Bobrov 1994; Bell et al. 2001; Bremer et al. 2001; Zhang et 1957; Mabberley 1997; Mayer 2016). The genus Succi al. 2003; Caputo et al. 2004; Bell & Donoghue 2005; sella contains five currently accepted species. Three of Avino et al. 2009; Carlson et al. 2009, etc.). Earlier them (S. carvalhoana (Mariz) Baksay, S. microcephala researchers subdivided Dipsacaceae mainly into three (Willk.) G. Beck, and S. andreae-molinae Pajarón tribes: Knautieae Janchen, Dipsaceae Rchb., and & Escudero) are endemic to the Iberian Peninsula Scabioseae DC. Succisa and Succisella were usually (Amich et al. 2004). Succisella petteri (Kerner & Murb.) assigned to Scabioseae, together with Scabiosa ��L.�� (��Bo- G. Beck is endemic to the Dinara Planina (Bosnia and brov 1957; Kotov 1961, etc.). According to results of Croatia) and S. inﬂexa (Kluk) G. Beck is distributed molecular phylogenetic studies, Succisa and Succisella in southern and eastern parts of Europe, from Italy are sister taxa in the ITS and combined analyses, and to Baltic states in the north and the Caucasus in the only in cpDNA analyses, Succisella was resolved as south. In other regions, S. inﬂexa is considered to be VARIABILITY, TAXONOMY AND PHYLOGENY TAXONOMY VARIABILITY,

alien and naturalized (Amich et al. 2004; Mabberley Biolar, ×700), the pollen was acetolyzed following 1997; Mayer 2016). Two species, Succisa pratensis Erdtman (1952). For size determinations, 20 measure- and Succisella inﬂexa, are native members the flora of ments were taken along the polar and equatorial axes for Ukraine and adjacent countries (Bobrov 1957; Kotov each species. Permanent preparations of pollen grains of 1961; Mosyakin & Fedoronchuk 1999). Succisa and Succisella are deposited in the Palynotheca at Pollen grains of Succisa pratensis were earlier the National Herbarium of Ukraine (KW). For scanning described using light microscopy (Erdtman 1952; Vino electron microscopy (SEM, JSM-6060LA), pollen grains kurova 1959; Moore & Webb 1983). The PalDat electro were treated with 96%-ethanol, then the samples were nic database contains information on the pollen morpho sputter-coated with gold and investigated at the Center logy of S. pratensis under scanning electron microscopy of Electron Microscopy of the M.G. Kholodny Institute (Halbritter & Svojtka 2016). Pollen grains of S. pratensis of Botany. The micrographs were minimally edited with were also studied using both light and scanning electron Adobe Photoshop 6.0 to enhance images. microscopy (Clarke & Jones 1981). The morphological Terminology used in descriptions of pollen grains peculiarities of pollen grains of Iberian taxa Succisella mainly follows the glossary by Punt et al. (2007), with microcephala, S. andreae-molinae, and S. carvalhoana some necessary minor adjustments (Tsymbalyuk et al. were also investigated using both light and scanning 2018, 2019). Original and published data on quantita- electron microscopy (Amich et al. 2004). tive pollen characters representatives of Succiseae are Our main objective was to perform a more detailed summarized in Table 1. palynomorphological investigation of Ukrainian species Succisa pratensis and Succisella inﬂexa, to provide 3. Results assessment of the taxonomic significance of pollen 3.1. General description of pollen grains of Succiseae morphology of Succisa and Succisella based on both published and original data, and to explore possibilities Pollen grains in monads, radially symmetrical, of its uses in taxonomy. isopolar, tricolpate, prolate, rarely spheroidal or oblate- spheroidal, in polar view subcircular, rarely trilobate, 2. Materials and methods in equatorial view elliptical, rarely circular. Polar axis P=66.5-98.4 µm, equatorial axis E=65.2-98.4 µm. Colpi Pollen grains of Succisa and Succisella were sampled short, of variable width, 0.7-5.3 µm wide, 26.6-37.2 µm in the National Herbarium of Ukraine (KW – herbarium long, margin irregular with distinct, narrow margo, 0.7- of the M. G. Kholodny Institute of Botany, National 2.0 µm wide, with blunt or acute ends, colpus membrane Academy of Sciences of Ukraine, Kyiv, Ukraine). Data smooth or granulate, often ruptured during acetolysis. on the studied specimens are cited exactly according to Exine 6.6-10.6 µm thick, unevenly thickened, rarely the label information, in English translation. on apocolpia thicker. Columellae distinct, short, occa The methods used in the present study are essentially sionally longer in apocolpium. Exine sculpture echinate- the same as those we used earlier (see Mosyakin & microechinate. Echinae conical with straight sides and Tsymbalyuk 2015a, 2015b, 2017). Pollen morphology acute apex, 0.9-1.5 µm high, 1.0-1.3 µm wide at base. was studied using both light microscopy and scanning Microechinae small, more numerous than echinae, 0.2- electron microscopy. For light microscopy studies (LM, 0.3 µm high, 0.3-0.4 µm wide at base. Exine surface

Table 1. Morphometric comparison of pollen grains

Polar axis, P Equatorial axis, Colpi Colpi width Margo Exine Taxon (µm) E(µm) length(µm) (µm) (µm) (µm) Succisa pratensis 68(77)-84(95) 69(75)-79(98) 26-32 3-7 1.5-2.5 8-11 (Clarke & Jones 1981) Succisa pratensis 73.1-98.4 66.5-98.4 27.9-37.2 2.0-5.3 1.3-2.0 6.6-10.6 (original data) Succisella infexa 66.5-79.8 65.2-79.8 26.6-29.3 0.7-4.0 0.7-1.6 6.6-9.3 (original data) Succisella carvalhoana 63.60±3.52 65.71±2.71 ld ld ld ld (Amich et al. 2004) Succisella microcephala 64.83±2.98 66.93±2.65 ld ld ld ld (Amich et al. 2004) Succisella andreae-molinae 64.11±3.10 66.27±2.89 ld ld ld ld (Amich et al. 2004)

Explanation: ld – lack of data Biodiv. Res. Conserv. 55: 1-6, 2019 3 between echinae and microechinae rugulate or rugula fused into groups, branched above, arranged more or te-perforate. Colpus membrane with irregular granules less regularly, rarely on apocolpia longer. Endexine and short echinae. unevenly thickened. Echinae indistinct on the surface, distinct on the edges, sparsely distributed. Columellae 3.2. Descriptions of pollen grains distinct, circular, elongated or angular, at the base in Genus Succisa Haller LO analysis, densely distributed. Succisa pratensis Moench. (Fig. 1a-c; Fig. 2a-d) S E M . Exine sculpture echinate-microechinate. LM. Pollen grains tricolpate, prolate, rarely spheroidal, Echinae conical with straight sides and acute apex, 1.3- often with slightly undulating margin, in polar view 1.5 µm high, 1.2-1.3 µm wide at base. Microechinae subcircular, rarely trilobate, in equatorial view elliptical, small, more numerous than echinae, 0.2-0.3 µm high, rarely circular. P=73.1-98.4 µm, E=66.5-98.4 µm. Colpi 0.3-0.4 µm wide at base. Exine surface between echinae short, 2.0-5.3 µm wide, 27.9-37.2 µm long, margin and microechinae rugulate-perforate. Colpus membrane irregular with distinct, narrow margo, 1.3-2.0 µm wide, with irregular granules and short echinae. with blunt or acute ends, colpus membrane smooth or Specimens investigated: 1. Ukraine, Zhyto granulate, often ruptured during acetolysis. Exine 6.6- myr Region. Volodarsk-Volynsky District. St. Turchan- 10.6 µm thick, unevenly thickened, rarely on apocolpia ka, pasture in the birch-alder forest. ��30.07.1949��.�� A.�� Za- thicker. Tectum 3-4 times thinner than infratectum. pyatova (KW). 2. Ukraine, near Vinnitsa [Vinnytsya], Columellae distinct, short, of variable thickness, often wet meadow north of the railway in the direction of

a b

c d

e f

Fig. 1. Pollen grains of Succisa pratensis and Succisella infexa (SEM) Explanations: a-c – S. pratensis, d-f – S. infexa, a, e – polar view, b, f – colpi membranes with irregular granules and short echinae, c, d – exine sculpture 4 Zoya M. Tsymbalyuk et al. Taxonomic significance ofSuccisa and Succisella pollen morphology

Sosenke (Kalinovka). 24.VIII 1927. K.S. Yankovsky Specimens investigated: 1. Ukraine, Volyn' (KW). 3. Ukraine, Chernihiv Region, Lyubech District, Region, Kovel District, v.[village] Ugli, floodplain of Lyubech village, floodplain of the Dnipro [Dnieper] the Stohod [Stokhid] River, peat meadows. 3. VIII River. 28.VII 1952. D. Afanasyev (KW). 2007. T.L. Andriyenko. 083618 (KW). 2. Ukraine, Lviv Region, Kamenka-Buz'ka District, northwestern Genus Succisella G. Beck outskirts of Tishitsa village, right bank of the West- Succisella infexa (Kluk) G. Beck ern Bug River, wet meadow. ��20.07.1999. O.T. Ku- (Fig. 1d-f; Fig. 2e-h) zyarin. 056940 (KW). 3. Belarus, Kamenyuki village, LM. Pollen grains tricolpate, prolate, rarely oblate- Kamenetsk District, Brest Region. July 26, 1972. O. spheroidal or spheroidal, in polar view subcircular, rarely Dubovik (KW). trilobate, in equatorial view elliptical, rarely circular. P=66.5-79.8 µm, E=65.2-79.8 µm. Colpi short, 0.7-4.0 4. Discussion µm wide, 26.6-29.3 µm long, margin irregular with distinct, narrow margo, 0.7-1.6 µm wide, with acute or In general, our data are in good agreement with sometimes blunt ends, colpus membrane smooth and the results of previous studies (Clarke & Jones 1981; granulate, often ruptured during acetolysis. Exine 6.6-9.3 Amich et al. 2004). In particular, Amich et al. (2004) µm thick, unevenly thickened. Tectum 4 times thinner provided the following information about pollen grains than infratectum. Columellae distinct, short, arranged of Succisella: “The pollen of the Iberian Peninsula spe- more or less regularly. Endexine unevenly thickened. cies of Succisella (...) is tricolpate (...). No differences Echinae indistinct on the surface and at the edges, very were seen between the studied taxa. (...) In all cases, the sparsely distributed. Columellae distinct, circular, elon- pollen is oblate-spheroid, almost spheroid, (Figs 13-15) gated and angular at the base in LO analysis, densely with a weakly striated exine (Figs 19-21), covered in distributed. small, sparse spines, the largest of which are 0.8-1 mm S E M . Exine sculpture echinate-microechinate. in S. microcephala and S. andreae-molinae, and the Echinae conical with straight sides and acute apex, 0.9- smallest 0.2-0.3 mm in S. carvalhoana (Figs 16-18). 1.1 µm high, 1.0-1.2 µm wide at base. Microechinae These palynological characteristics reveal the primitive small, more numerous than echinae, 0.2 µm high, nature of the genus within the Dipsacaceae” (Amich et 0.3 µm wide at base. Exine surface between echinae al. 2004, p. 355). and microechinae rugulate. Colpus membrane with Pollen grains of Succisella infexa are similar to irregular granules and short echinae. those of the Iberian Succisella in their pollen type, size,

a b c d

e f g h

Fig. 2. Pollen grains of Succisa pratensis and Succisella infexa (LM) Explanations: a-d – S. pratensis, e-h – S. infexa, a, e – equatorial view (× 400), b, f – polar view (× 400), c, g – exine (× 700), d, h – colpi (× 700); scale bars: a-h – 10 µm Biodiv. Res. Conserv. 55: 1-6, 2019 5 and exine sculpture. Unfortunately, the illustrations and supports the phylogenetic hypothesis (Caputo et al. characters reported by Amich et al. (2004) are insuf- 2004; Carlson et al. 2009) that the ‛Knautieae’ group is ficient for a more detailed comparative analysis. more closely related to Dipsacus and Cephalaria than Analysis of our original palynomorphological data to Succisa and Succisella. The palynomorphological and literature records demonstrated that pollen grains characters in Succiseae can be probably viewed as of Succisa and Succisella representatives are characte ancestral, or, at least, close to ancestral, or primary for rized by the tricolpate type of apertures with echinate- those registered in other members of Dipsacaceae sensu microechinate sculpture (see also Clarke & Jones 1981; stricto. Amich et al. 2004; Mayer & Ehrendorfer 2013, and Acknowledgements. The authors express their gratitude to compare to original data reported here); however, they Natalia M. Shyian, Head Curator of the National Herbarium differ in their size, colpi structure, and exine structure of Ukraine (KW; M.G. Kholodny Institute of Botany, Na- and sculpture. Pollen grains of Succisa are larger in their tional Academy of Sciences of Ukraine), for her cooperation size, with wider and longer colpi, wider margo, and and assistance in extracting pollen samples from herbarium thicker exine, as compared to pollen of Succisella. The specimens at KW. Kind help and cooperation of Dmytro O. exine sculpture in both taxa is echinate-microechinate, Klymchuk, Head of the Center of Electron Microscopy, and but in pollen grains of Succisa echinae are larger than Vitaliy I. Sapsay, SEM operator (M. G. Kholodny Institute those in Succisella (Clarke & Jones 1981; Amich et al. of Botany, National Academy of Sciences of Ukraine), are greatly appreciated. We also thank Veronika Mayer (Division 2004, and original data). The species-specific features of Structural and Functional Botany, University of Vienna, mentioned above are mainly visible under a light mi- Vienna, Austria) for providing several important references. croscope (see LM descriptions above) and, therefore, The authors are grateful to anonymous reviewers and the LM can be used in performing pollen analysis. editorial staff of the journal for their valuable comments Thus, available palynomorphological data are well and suggestions. consistent with molecular phylogenetic evidence (Avino et al. 2009; Carlson et al.�� 2009). Data on pollen morphology also confirm the taxonomic circumscription of Author Contributions Succiseae as outlined by Mayer (2016). Pollen grains Research concept and design: Z. M. Tsymbalyuk, S. L. Mosyakin in Succiseae are characterized by a tricolpate aperture Acquisition and/or assembly of data: Z. M. Tsymbalyuk, L. type (see details above). In contrast, representatives of M. Nitsenko Knautia L., Dipsacus L., and Cephalaria Schrad. ex Data analysis and interpretation: Z. M. Tsymbalyuk Roem. & Schult. usually have a triporate aperture type Drafting the article: Z. M. Tsymbalyuk, L. M. Nitsenko (Clarke & Jones 1981; Tsymbalyuk et al. 2018, 2019, Critical revision: S. L. Mosyakin and references therein). Palynological evidence, thus, Final approval: S. L. Mosyakin, Z. M. Tsymbalyuk

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